PROJECT SUMMARY/ABSTRACT Erythropoiesis is a dynamic process governed by quantitative changes in the relative levels of transcription fac- tors (TFs), their specific isoforms and post-translational modifications (PTMs). Due to the current paucity of quantitative data on the proteins that constitute the transcriptional regulatory network, current models of eryth- ropoiesis are based primarily on mRNA measurements and do not typically consider changes in the protein levels of specific TFs, their isoforms or PTMs. This significantly limits the understanding of erythropoiesis and other transcriptionally regulated processes such as ss-globin expression, ultimately impinging on the capacity to correct hemoglobin disorders. The long-term goal is to decipher the transcriptional network that controls eryth- ropoiesis in health and disease. The objective of this proposal is to build a network model of erythropoiesis based on dynamic changes in TF protein levels during erythroid differentiation of human hematopoietic stem/progenitor cells (HSPCs). The central hypothesis is that the relative protein levels of TFs is a critical pa- rameter in the establishment of proper gene expression programs at each stage of differentiation, and that erythropoiesis is driven by graded changes in the relative amounts of specific combinations of TFs. The ra- tionale is that integration of the dynamic and quantitative nature of the proteome into the transcriptional net- work of erythropoiesis will result in a model with improved predictive power which will serve as a benchmark for healthy erythropoiesis against which to compare erythroid-related disease states, and will facilitate the identifi- cation of pharmacological agents to restore normal erythropoiesis. Two specific aims have been designed: 1) Model the erythropoiesis transcriptional network based on measurements of dynamic changes in the protein levels of transcription factors; and 2) Identify novel changes in abundance of nuclear proteins and phosphopro- teins during erythropoiesis. Under the first aim, a novel targeted mass spectrometry approach developed by the applicants will be used to measure absolute levels of TFs at multiple stages during ex vivo erythropoiesis of HSPCs derived from healthy donors. Under the second aim, an unbiased proteomic approach will be used to identify previously unappreciated proteins that undergo quantitative changes in their levels and/or phosphoryla- tion status during ex vivo erythropoiesis of HSPCs from healthy donors. The approach is innovative because it uses novel mass spectrometry approaches to systematically identify and quantify TFs that regulate erythropoi- esis in primary human cells. The proposed research is significant because it will illuminate complex regulatory processes that control erythropoiesis. Ultimately, such knowledge has the potential to guide the design of new therapeutics to re-establish proper ss-globin expression in ss-thalassemic patients.